T cell costimulatory pathways play key roles in regulating T cell activation and tolerance. Recent genetic studies suggest that costimulatory molecules control genetic susceptibility to many human autoimmune diseases. Project 4 will dissect the functional roles of CTLA-4 and the recently identified costimulatory pathway consisting of the costimulatory receptor CD226, the coinhibitory receptor TIGIT and their ligand PVR, in regulating the balance between effector versus regulatory T cell responses, and development of autoimmunity. This focus is driven by human genetic studies of Drs. Todd, Wicker (Project 1), and Dr. Hafler (Project 2) which have identified candidate causal variants in CTLA-4 and CD226 associated with risk of developing autoimmune diseases, together with our studies of the immunoregulatory functions of CTLA-4, and our finding of CD28-dependent upregulation and stabilization of CD226 expression. We will test our hypothesis that T cell costimulatory pathways play critical roles in regulating the balance between pathogenic and protective T cell responses. To test this hypothesis, we propose the following Specific Aims: 1. To investigate how CTLA-4 controls the balance between effector and regulatory T cells. We will use CTLA-4 conditional knockout mice (KO) to analyze how CTLA-4 controls effector and regulatory T cell development and function. 2. To investigate the interplay between the CD28/CTLA-4 with CD226/TIGIT pathways. We will investigate how CD28 and CTLA-4 regulate expression of CD226 isoforms as well as CD226 protein, since our preliminary data indicate that CD28 may differentially regulate CD226 isoform expression. We also will test the hypothesis that dysregulated CD226 expression may contribute to the phenotype of CTLA-4K0 mice, since elimination of CD28 abrogates the CD226 phenotype. In complementary studies, we will investigate the contribution of CD28 to the activated phenotype of TIGIT-/- mice. 3. To investigate the functions of PVR, focusing on mechanisms by which TIGIT/PVR interactions inhibit T cell responses using TIGIT-Fc and PVR KO mice. Our goals are to understand how the CD28/CTLA-4 and CD226/TIGIT/PVR interactions regulate T cell tolerance and autoimmunity.